CFTR channels are tightly regulated by their level of phosphorylation, which is determined by the activities of protein kinases and phosphatases. We hypothesize that in vivo regulation of CFTR is mediated, at least in part, by a robust, membrane-bound phosphatase that also causes channels to run down in excised patches. Despite their obvious importance, the phosphatases acting on CFTR have not been identified at the molecular level and the factors modulating their control of CFTR activity remain poorly understood. The longterm objectives of this project are to identify the membrane-bound phosphatase and to understand the regulation of CFTR by phosphatases generally. Functional studies strongly suggest it resembles FF20; however, PP2C is a protein family that includes multiple isoforms and splice variants. Moreover, our recent mass spectrometry data suggest that it may be a new type of PP2C with little (. 30 %) homology to known isoforms. We previously showed that CFTR is specifically associated with a PP2C-like phosphatase that can be affinity purified using tagged CFTR. The first specific aim of this proposal is to verify that the protein we have recently identified by 2D gel electrophoresis and mass spectrometry is a CFTR phosphatase. The second specific aim is to study the basic enzymatic properties of the native and recombinant phosphatase (i.e. its kinetics, substrate specificity) in order to understand the mechanism of CFTR deactivation. The third specific aim will explore the interaction between PP2C and CFTR to determine if it is direct or mediated by an AKAP-like protein, identify region(s) on both CFTR and the phosphatase that are necessary for association within the regulatory complex. The phosphatase cDNA will be used in a yeast two-hybrid screen for scaffold proteins that may anchor it to CFTR. The fourth specific aim is to investigate the regulation of CFTR dephosphorylation at the level of the phosphatase. This work should provide new insights into the physiological control of CFTR and its interactions with regulatory proteins, and may be useful as a drug target in the future development of phosphatase inhibitors and activators as potential pharmacotherapies in cystic fibrosis and secretory diarrhea.